Distinct antiviral activities of IFNφ1 and IFNφ4 in zebrafish.

Interferons (IFNs) are a group of secreted cytokines that play a crucial role in antiviral immunity. Type I IFNs display functional disparities. In teleosts, type I IFNs are categorized into two subgroups containing one or two pairs of disulfide bonds. However, their functional differences have not been fully elucidated. In this study, we comparatively characterized the antiviral activities of zebrafish IFNφ1 and IFNφ4 belonging to the group I type I IFNs. It was found that ifnφ1 and ifnφ4 were differentially modulated during viral infection. Although both IFNφ1 and IFNφ4 activated JAK-STAT signaling pathway via CRFB1/CRFB5 receptor complex, IFNφ4 was less potent in inducing phosphorylation of STAT1a, STAT1b and STAT2 and the expression of antiviral genes than IFNφ1, thereby conferring weaker antiviral resistance of target cells. Taken together, our results provide insights into the functional divergence of type I IFNs in lower vertebrates.

The Biological Functions and Intestinal Inflammation Regulation of IL-21 in Grass Carp (Ctenopharyngodon idella) during Infection with Aeromonas hydrophila.

Interleukin (IL) 21 is a pleiotropic cytokine that plays an important role in regulating innate and adaptive immune responses. In fish, the biological functions and cell source of IL-21 remain largely unknown. In this study, we performed qRT-PCR, Western blotting and immunofluorescent microscopy to examine the expression of IL-21 at the mRNA and protein levels. We found that il21 expression was induced in the primary head kidney leukocytes of grass carp (Ctenopharyngodon idella) by heat-inactivated Aeromonas hydrophila (A. hydrophila) and LPS and in tissues after infection with A. hydrophila. Recombinant IL-21 protein produced in the CHO-S cells was effective in elevating the expression of antibacterial genes, including ß-defensin and lysozyme, and, interestingly, inhibited the NF-κB signaling pathway. Furthermore, we investigated the response of the IL-21 expressing cells to A. hydrophila infection. Immunofluorescent assay showed that IL-21 protein was detected in the CD3γ/δ T cells and was markedly accumulated in the anterior, middle and posterior intestine. Collectively, the results indicate that IL-21 plays an important role in regulating the intestinal inflammation induced by bacterial infection in grass carp.

DDX5 inhibits type I IFN production by promoting degradation of TBK1 and disrupting formation of TBK1 - TRAF3 complex.

DExD/H-box helicase (DDX) 5 belongs to the DExD/H-box helicase family. DDX family members play differential roles in the regulation of innate antiviral immune response. However, whether DDX5 is involved in antiviral immunity remains unclear. In this study, we found that DDX5 serves as a negative regulator of type I interferon (IFN) response. Overexpression of DDX5 inhibited IFN production induced by Spring viremia of carp virus (SVCV) and poly(I:C) and enhanced virus replication by targeting key elements of the RLR signaling pathway (MAVS, MITA, TBK1, IRF3 and IRF7). Mechanistically, DDX5 directly interacted with TBK1 to promote its autophagy-mediated degradation. Moreover, DDX5 was shown to block the interaction between TRAF3 and TBK1, hence preventing nuclear translocation of IRF3. Together, these data shed light on the roles of DDX5 in regulating IFN response.

CBFß is induced by spring viremia of carp virus and promotes virus replication in zebrafish.

The core binding factor subunit beta (CBFß) is a transcription factor that forms a complex with virial proteins to promote viral infection. In this study, we identified a CBFß homolog from zebrafish (zfCBFß) and characterized the biological activity. The deduced zfCBFß protein was highly similar to orthologs from other species. The zfcbfß gene was constitutively expressed in tissues and was induced in immune tissues after infection with spring viremia carp virus (SVCV) and stimulation with poly(I:C). Interestingly, zfcbfß is not induced by type I interferons. Overexpression of zfcbfß induced tnfα expression but inhibited isg15 expression. Also, overexpression of zfcbfß significantly increased SVCV titer in the EPC cells. Co-immunoprecipitation assay revealed that zfCBFß interacts with SVCV phosphoprotein (SVCVP) and host p53, resulting in the increased stability of zfCBFß. Our results provide evidence that CBFß is targeted by virus to suppress host antiviral response.

Zebrafish SETD3 mediated ubiquitination of phosphoprotein limits spring viremia of carp virus infection.

Lysine methylation is a post-translational modification of histone and non-histone proteins and affects numerous cellular processes. The actin histidine methyltransferase SET domain containing 3 (SETD3) is a member of the protein lysine methyltransferase (PKMT) family which catalyse the addition of methyl groups to lysine residues. However, the role of SETD3 in virus-mediated innate immune responses has rarely been investigated. In this study, zebrafish SETD3 was shown to be induced by poly(I:C) and spring viremia of carp virus (SVCV) and inhibited virus infection. Further, it was found that SETD3 directly interacted with SVCV phosphoprotein (SVCV P) in the cytoplasm of EPC cells, initiating ubiquitination to degrade the SVCV P protein via proteasomal pathway. Interestingly, mutants lacking the SET and RSB domains were able to promote degradation of SVCV P, indicating that they are not required for SETD3 mediated degradation of SVCV P. Taken together, our study demonstrates that SETD3 is an antiviral factor which limits virus replication by promoting ubiquitination of viral phosphoprotein and subsequent protein degradation.

TL1A induces apoptosis via DR3 in grass carp (Ctenopharyngodon idella).

Tumor necrosis factor like ligand 1A (TL1A), a member of TNF superfamily, regulates inflammatory response and immune defense. TL1A homologues have recently been discovered in fish, but their functions have not been studied. In this study, a TL1A homologue was identified in grass carp (Ctenopharyngodon idella) and its bioactivities were investigated. The grass carp tl1a (Citl1a) gene was constitutively expressed in tissues, with the highest expression detected in the liver. It was upregulated in response to infection with Aeromonas hydrophila. The recombinant CiTL1A was produced in bacteria and was shown to stimulate the expression of il1ß, tnfα, caspase 8 and ifnγ in the primary head kidney leucocytes. In addition, co-immunoprecipitation assay revealed that CiTL1A interacted with DR3 and induced apoptosis via activation of DR3. The results demonstrate that TL1A regulates inflammation and apoptosis and is involved in the immune defense against bacterial infection in fish.

IL-4/13 expressing CD3γ/δ+ T cells regulate mucosal immunity in response to Flavobacterium columnare infection in grass carp.

Interleukin (IL) 4 and 13 are signature cytokines orchestrating Th2 immune response. Teleost fish have two homologs, termed IL-4/13A and IL-4/13B, and have been functionally characterized. However, what cells express IL-4/13A and IL-4/13B has not been investigated in fish. In this work, the recombinant IL-4/13A and IL-4/13B proteins of grass carp (Ctenopharyngodon idella) were produced in the Escherichia coli (E. coli) cells and purified. Monoclonal antibodies (mAbs) against the recombinant CiIL-4/13A and CiIL-4/13B proteins were prepared and characterized. Western blotting analysis showed that the CiIL-4/13A and CiIL-4/13B mAbs could specifically recognize the recombinant proteins expressed in the E. coli cells and HEK293T cells and did not cross-react with each other. Confocal microscopy revealed that the CiIL-4/13A+ and CiIL-4/13B+ cells were present in the gills, intestine and spleen and could be upregulated in fish infected with Flavobacterium columnare (F. columnare). Interestingly, the cells expressing CiIL-4/13A and CiIL-4/13B were mostly CD3γ/δ+ cells. The CD3γ/δ+/IL-4/13A+ and CD3γ/δ+/IL-4/13B+ cells were significantly upregulated in the gill filaments and the intestinal mucosa after F. columnare infection. Our results imply that the CD3γ/δ+/IL-4/13A+ and CD3γ/δ+/IL-4/13B+ cells are important for homeostasis and the regulation of mucosal immunity.

The expanded ISG12 family in zebrafish: ISG12.1 suppresses virus replication via targeting viral phosphoprotein.

In mammals, interferon (IFN)-stimulated genes (ISGs) play important roles in restricting the replication of viruses. However, the functions of many ISGs have not been investigated in fish. In this study, eight isg12 homologs (termed isg12.1-8) were identified in zebrafish and all contain a typical ISG12 family domain rich of hydrophobic amino acid residues. Isg12.1-7 were significantly induced in the ZF4 cells by poly(I:C) and IFNφ1, and in the kidney and spleen after infection with spring viremia of carp virus (SVCV). In the EPC cells, overexpression of isg12.1 inhibited SVCV replication. Further, it was found that zebrafish ISG12.1 interacted with SVCV phosphoprotein (SVCV-P) and promoted SVCV-P degradation which could be attenuated by 3-MA and CQ (autophagy inhibitors). Our results indicate that zebrafish ISG12.1 restricts viral replication by targeting viral phosphoprotein for degradation.

IL-27 suppresses spring viremia of carp virus replication in zebrafish.

Interleukin (IL) 27 is a member of the IL-12 family and is a heterodimeric cytokine composed of IL-27A and Epstein-Barr virus-induced 3 (EBI3). It plays an important role in regulating inflammation and cancer progression. IL-27A not only functions by dimerizing with EBI3 but also acts alone. Here, we report that IL-27A and EBI3 suppress spring viremia of carp virus (SVCV) replication in zebrafish. Expression analysis reveals that il-27a and ebi3 were significantly upregulated in the ZF4 cells by SVCV and poly(I:C), and in the zebrafish caudal fin (ZFIN) cells overexpressed with SVCV genes. Interestingly, il-27a and ebi3 were not modulated by IFNφ1, indicating that they are not IFN stimulated genes (ISGs). Furthermore, overexpression of IL-27A and EBI3 alone inhibited SVCV replication in the EPC cells, but less potent than co-expression of IL-27A and EBI3. Intriguingly, IL-27A could not induce the expression of irf3, ifn, isg15 and mx1. Taken together, our results demonstrate that IL-27A and EBI3 activate innate antiviral response in an IFN independent manner in zebrafish.

The Meteorin-like cytokine is upregulated in grass carp after infection with Aeromonas hydrophila.

Meteorin-like (Metrnl) is a novel immune regulatory factor or adipokine which is mainly produced by activated macrophages. In teleost fish, two homologs are present. In this study, monoclonal antibodies were prepared against recombinant grass carp (Ctenopharyngodon idella, Ci) Metrnl-a in mice and characterized by Western blotting, flow cytometry and immunofluorescent microscopy. In grass carp infected with Aeromonus hydrophila (A. hydrophila), the cells expressing CiMetrnl-a markedly increased in the gills, head kidney and intestine. In the inflamed intestine caused by A. hydrophila infection, the CiMetrnl-a producing cells were detected mainly in the mucosal layer of anterior, middle and posterior segments. Consistently, qRT-PCR analysis showed that the mRNA expression of CiMetrnl-a was markedly induced. Our results suggest that CiMetrnl-a is involved in regulating intestine inflammation caused by bacterial infection.

IRF2 Cooperates with Phosphoprotein of Spring Viremia of Carp Virus to Suppress Antiviral Response in Zebrafish.

IFN regulatory factor (IRF) 2 belongs to the IRF1 subfamily, and its functions are not yet fully understood. In this study, we showed that IRF2a was a negative regulator of the interferon (IFN) response induced by spring viremia of carp virus (SVCV). Irf2a-/- knockout zebrafish were less susceptible to SVCV than wild-type fish. Transcriptomic analysis reveals that differentially expressed genes (DEGs) in the irf2a-/- and irf2a+/+ cells derived caudal fins were mainly involved in cytokine-cytokine receptor interaction, mitogen-activated protein kinase (MAPK) signaling pathway, and transforming growth factor-beta (TGF-beta) signaling pathway. Interestingly, the basal expression levels of interferon stimulating genes (ISGs), including pkz, mx, apol, and stat1 were higher in the irf2a-/- cells than irf2a+/+ cells, suggesting that they may contribute to the increased viral resistance of the irf2a-/- cells. Overexpression of IRF2a inhibited the activation of ifnφ1 and ifnφ3 induced by SVCV and poly(I:C) in the epithelioma papulosum cyprini (EPC) cells. Further, it was found that SVCV phosphoprotein (SVCV-P) could interact with IRF2a to promote IRF2a nuclear translocation and protein stability via suppressing K48-linked ubiquitination of IRF2a. Both IRF2a and SVCV-P not only destabilized STAT1a but reduced its translocation into the nucleus. Our work demonstrates that IRF2a cooperates with SVCV-P to suppress host antiviral response against viral infection in zebrafish. IMPORTANCE Interferon regulatory factors (IRFs) are central in the regulation of interferon-mediated antiviral immunity. Here, we reported that IRF2a suppressed interferon response and promoted virus replication in zebrafish. The suppressive effects were enhanced by the phosphoprotein of the spring viremia of carp virus (SVCV) via inhibition of K48-linked ubiquitination of IRF2a. IRF2a and SVCV phosphoprotein cooperated to degrade STAT1 and block its nuclear translocation. Our work demonstrated that IRFs and STATs were targeted by the virus through posttranslational modifications to repress interferon-mediated antiviral response in lower vertebrates.

Essential Oils From Citrus unshiu Marc. Effectively Kill Aeromonas hydrophila by Destroying Cell Membrane Integrity, Influencing Cell Potential, and Leaking Intracellular Substances.

Aeromonas hydrophila is one of the important pathogenic bacteria in aquaculture causing serious losses every year. Essential oils are usually used as natural antimicrobial agents to reduce or replace the use of antibiotics. The aim of this study was to evaluate the antibacterial activity and explore the mechanisms of essential oil from satsuma mandarin (Citrus unshiu Marc.) (SMEO) against A. hydrophila. The results of the gas chromatography-mass spectrometer demonstrated that SMEO contains 79 chemical components with the highest proportion of limonene (70.22%). SMEO exhibited strong antibacterial activity against A. hydrophila in vitro, the diameter of the inhibition zone was 31.22 ± 0.46 mm, and the MIC and MBC values were all 1% (v/v). Intracellular material release, scanning electron microscopy (SEM), transmission electron microscopy (TEM), and flow cytometry analysis revealed the dynamic antibacterial process of SMEO, the morphological changes of bacterial cells, and the leakage process of intracellular components. These results demonstrated that SMEO disrupted the extracellular membrane permeability. Our study demonstrated that SEMO has the potential to be used to control and prevent A. hydrophila infections in aquaculture.

PLAAT1 promotes p53 degradation via autophagy-lysosome pathway in zebrafish.

PLAAT1 belongs to the PLAAT family and plays regulatory roles in cell growth, tumor suppression and phospholipid metabolism. However, whether PLAAT1 is involved in p53 mediated signaling has not been investigated. Here, we report that PLAAT1 promotes degradation of p53 in zebrafish. We found that the plaat1 gene was constitutively expressed in tissues including liver, kidney, spleen, intestine, eye and brain, with relative higher expression levels detected in the brain and eye. Overexpression of plaat1 led to inhibition of p53 and tnfα mRNA expression. Furthermore, it was shown that PLAAT1 interacted with p53 to facilitate p53 degradation via autophagy-lysosome dependent pathway. Our work indicates that PLAAT1 is involved in the interplay between p53 mediated cellular responses and autophagy.

Structural and Functional Analyses of Type I IFNa Shed Light Into Its Interaction With Multiple Receptors in Fish.

Teleost type I interferons (IFNs) are categorized into group I and II subgroups that bind to distinct receptors to activate antiviral responses. However, the interaction between ifn ligands and receptors has not fully been understood. In this study, the crystal structure of grass carp [Ctenopharyngodon idella (Ci)] IFNa has been solved at 1.58Å and consists of six helices. The CiIFNa displays a typical structure of type I IFNs with a straight helix F and lacks a helix element in the AB loop. Superposition modeling identified several key residues involved in the interaction with receptors. It was found that CiIFNa bound to cytokine receptor family B (CRFB) 1, CRFB2, and CRFB5, and the three receptors could form heterodimeric receptor complexes. Furthermore, mutation of Leu27, Glu103, Lys117, and His165 markedly decreased the phosphorylation of signal transducer and activator of transcription (STAT) 1a induced by CiIFNa in the Epithelioma papulosum cyprini (EPC) cells, and Glu103 was shown to be required for the CiIFNa-activated antiviral activity. Interestingly, wild-type and mutant CiIFNa proteins did not alter the phosphorylation levels of STAT1b. Our results demonstrate that fish type I IFNs, although structurally conserved, interact with the receptors in a manner that may differ from mammalian homologs.

Molecular identification of Klebsiella pneumoniae and expression of immune genes in infected spotted gar Lepisosteus oculatus.

Spotted gar (Lepisosteus oculatus) is a primitive ray-finned fish which has not undergone the third round whole genome duplication and commonly used as a model to study the evolution of immune genes. In this study, a pathogenic strain of Klebsiella pneumoniae (termed KPY01) was isolated from a diseased spotted gar, based on the Gram-stain and phylogenetic analysis of the 16S rDNA and khe genes. Further, the virulence genes and drug resistance genes were determined and drug sensitivity tests were performed to explore the virulence and drug resistance of the KPY01. Putative biosynthetic gene clusters (BGCs) for the biosynthesis of secondary metabolites were predicted using the anti-SMASH5.0 online genome mining platform. Histopathological analysis revealed that the immune cells were significantly decreased in the white pulp of spleen of fish infected with K. pneumonia and tissue inflammation became apparent. Besides, the expression of cytokines including interleukin (il) -8, il-10, il-12a, il-18 and interferon γ (ifn-γ) were shown to be modulated in the spleen, gills and kidney. Our work provides useful information for further investigation on the virulence of K. pneumoniae and host immune responses to K. pneumoniae infection in fish.

CCL19 variants mediate chemotactic response via CCR7 in grass carp Ctenopharyngodon idella.

CC chemokine ligand 19 (CCL19) plays a key role in the regulation of immune responses including homeostasis, inflammation, and immune tolerance. In this study, two variants of CCL19 homologues (CCL19a2 and CCL19b) and CCR7 were investigated in grass carp Ctenopharyngodon idella. The three genes were widely expressed in immune tissues and could be modulated by stimulation with LPS, PHA and poly(I:C), and infection with Flavobacterium columnare and grass carp reovirus. In an in vitro chemotaxis assay, the recombinant CCL19a2 and CCL19b were active to promote the migration of HEK293 T cells expressing CCR7 and leucocytes isolated from the gills, head kidney and spleen. Moreover, their chemotactive effects were validated in vivo. We found that the cells recruited by CCL19a2 and CCl19b are mainly monocytes/macrophages expressing high levels of IL-1ß, IFN-γ, colony stimulating factor 1 receptor (CSF1R) and MHC II. Our work suggests that CCL19a2 and CCl19b are involved in recruitment of antigen presenting cells in fish.

Identification and expression analysis of group II C-type lectin domain containing receptors in grass carp Ctenopharyngodon idella.

Group II C-type lectin domain (CTLD) containing receptors belong to a large family of pattern recognition receptors which mainly act on the innate immunity. They are structurally related and consist of a cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM) and a single extracellular CTLD. Although they have been described in teleost fish, their involvement in immune responses is not well understood. In this study, four immune-related lectin-like receptors (termed CiILLR1 and CiILLR5-7), belonging to the group II CTLD receptors, were identified in grass carp (Ctenopharyngodon idella). They contain a short cytoplasmic tail and a single CTLD in the extracellular region. The CiILLR1 has a WxHxxxxxY motif similar to the WxHxxxxY motif which is required for the recognition of ß-glucans by some of the group II CTLD containing lectins in mammals. Further, a modified QPD motif (EPD) known to be involved in binding to carbohydrate ligands is present in the CiILLR1, 5 and 6. However, CiILLR7 lacks these motifs. Expression analysis revealed that they were constitutively expressed in the head kidney and spleen. Moreover, CiILLR1, 5 and 6 could be up-regulated in the head kidney and spleen of fish after infection with Flavobacterium columnare and in the primary head kidney leukocytes by LPS and PHA. Expression of CiILLR1, CiILLR5 and CiILLR6 were mainly detected in the enriched lymphocytes whilst CiILLR7 was expressed in the enriched monocytes/macrophages. The results expand existing knowledge on the immune responses of the C-type lectin receptors in teleost fish.

Lipopolysaccharide-induced TNFα factor (LITAF) promotes inflammatory responses and activates apoptosis in zebrafish Danio rerio.

Lipopolysaccharide-induced TNFα factor (LITAF) is an important transcription factor which activates the transcription of TNFα and regulates cell apoptosis and inflammatory response. In the present study, a LITAF gene homologue was identified in zebrafish (Danio rerio) and was shown to be well conserved in the protein sequence, genomic organization and synteny with human LITAF. DrLITAF was constitutively expressed in tissues, with the highest expression detected in the gills. Its expression could be modulated by LPS, poly(I:C), and infection with Edwardsiella tarda, Aeromonus hydrophila and septicemia viremia of carp virus (SVCV). DrLITAF, when overexpressed, was shown to be located on the cellular membrane and nuclear membrane of HEK293T and ZF4 cells and was associated with the endoplasmic reticulum. Stimulation with LPS resulted in rapid translocation of DrLITAF into the nucleus. In addition, DrLITAF was able to induce cell apoptosis and the expression of caspase 3. The results demonstrate that DrLITAF is involved in the immune defence against bacterial and viral infection and plays a role in regulating inflammation and apoptosis.

Anti-listeria Activities of Linalool and Its Mechanism Revealed by Comparative Transcriptome Analysis.

Listeria monocytogenes, which causes serious foodborne infections and public health problems worldwide, is one of the most important foodborne pathogens. Linalool has been identified as an antimicrobial agent against some microorganism, but its mechanism of action is currently unclear. Here, we investigated the efficacy of linalool against L. monocytogenes while planktonic and as a biofilm and explored potential mechanisms of action. Linalool exhibited strong anti-listeria activity in the planktonic stage. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) observations revealed seven stages were classified of cells at microscopic level. Mesosome-like structures were observed for the first time in L. monocytogenes after linalool treatment. Linalool also showed significant anti-biofilm activity through both dispersal and killing of cells in the biofilm based on confocal scanning laser microscopy (CLSM) and SEM imaging, crystal violet staining, XTT and COMSTAT assays. Moreover, comparative transcriptome analysis demonstrated many potential mechanisms of action for linalool and some important pathways were screened out through the analysis of GO enrichment and KEGG. Our study provides evidence that linalool exhibits a strong antimicrobial activity against both the planktonic and biofilm forms of L. monocytogenes and gives insight into its mechanism of action.